# Genomic evolution and re-emergence of a multidrug-resistant Clostridioides difficile RT027 clone with reduced vancomycin susceptibility driving a prolonged hospital outbreak

## TL;DR

A multidrug-resistant Clostridioides difficile strain with reduced vancomycin susceptibility caused a 15-month hospital outbreak in Portugal, highlighting the need for stronger surveillance and infection control.

## Contribution

The study documents the genomic evolution and re-emergence of a high-risk RT027 C. difficile clone with expanded antimicrobial resistance and reduced vancomycin susceptibility.

## Key findings

- A multidrug-resistant RT027 C. difficile clone with reduced vancomycin susceptibility caused a 15-month hospital outbreak in Portugal.
- Genomic analysis revealed a rare VanR T115A mutation linked to reduced vancomycin susceptibility and close relatedness to U.S. isolates from 2016–2018.
- The outbreak was associated with a 25% recurrence rate and structural hospital limitations that facilitated transmission.

## Abstract

Following an extended period of declining prevalence of the epidemic Clostridioides difficile ribotype 027 (RT027) in Portugal, a genetically distinct, multidrug-resistant (MDR) RT027 strain with reduced susceptibility to vancomycin has emerged, causing a 15-month outbreak. This investigation provides epidemiological and genomic evidence for renewed circulation and evolutionary adaptation of this high-risk lineage. A comprehensive outbreak investigation was conducted in a tertiary-care hospital in northern Portugal between 2023 and 2025. Epidemiological and clinical data, antimicrobial exposures, and infection-control measures were analysed. Whole-genome sequencing (WGS) was performed to characterize the outbreak clone. Sixty-six RT027 C. difficile infection (CDI) cases were confirmed, with incidence peaking at 5.46 cases per 10,000 patient-bed days in April 2024. WGS revealed an unusual accumulation of AMR determinants conferring a broad MDR phenotype. Notably, all isolates harboured the VanR T115A substitution, a rare mutation previously linked to reduced vancomycin susceptibility, raising concerns regarding evolving antimicrobial tolerance within RT027. The close relatedness to 2016–2018 USA isolates suggests a recent emergence of this clone. Transmission was facilitated by structural constraints, limited isolation capacity and shared sanitary facilities. This prolonged outbreak documents the re-emergence and genomic evolution of a hypervirulent RT027 lineage, characterized by a concerning expansion of antimicrobial resistance and decreased vancomycin susceptibility and a high recurrence rate (25%). These findings highlight the ongoing adaptive potential of C. difficile under antimicrobial pressure and underscore the need for strengthened surveillance, genomic monitoring, and infection-prevention strategies to mitigate re-establishment of epidemic RT027 strains in Europe and beyond.